CN110713974A - Stem cell culture medium and preparation method thereof - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention discloses a stem cell culture medium and a preparation method thereof, wherein the stem cell culture medium comprises a matrix culture medium and an additive, the matrix culture medium is a DMEM/F12 liquid culture medium, and the additive components added into the matrix culture medium are ferric chloride, ammonia amide, glucose, a Chinese wolfberry extract, a tomato extract, vitamin C, insulin, an olive oil extract and a growth factor; the additive components used by the stem cell culture medium are ferric chloride, ammonia amide, glucose, a Chinese wolfberry extract, a tomato extract, vitamin C, insulin, an olive oil extract and growth factors, so that the stem cell culture medium does not contain animal serum and is convenient for separating stem cells at the later stage; the culture medium provided by the invention can further improve the culture efficiency of stem cells and reduce the use cost of the culture medium; improve the proliferation activity of the adipose-derived stem cells and the growth capacity of the adipose-derived stem cells.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a stem cell culture medium and a preparation method thereof.
Background
Stem cells are a type of pluripotent cells that have the ability to self-replicate. Under certain conditions, it can differentiate into a variety of functional cells. The stem cells are divided into embryonic stem cells and adult stem cells according to the development stage of the stem cells. The stem cells are classified into three categories according to their developmental potential: the stem cell culture medium comprises a stem cell culture medium body, a stem cell culture medium body and a stem cell culture medium body, wherein the stem cell culture medium body is prepared by adding a bovine serum base material, the bovine serum base material body is prepared by adding bovine serum base material, the bovine serum base material is prepared from various cells and various protein components, the protein components are difficult to determine, the separation of the stem cell in the later period is difficult, the hematopoietic stem cell is easy to cause interference, the proliferation capacity is poor and the like.
Disclosure of Invention
The purpose of the invention can be realized by the following technical scheme: a stem cell culture medium comprises a substrate culture medium and additives, wherein the substrate culture medium is a DMEM/F12 liquid culture medium, and the additives added into the substrate culture medium comprise ferric chloride, ammonia amide, glucose, a Chinese wolfberry extract, a tomato extract, vitamin C, insulin, an olive oil extract and growth factors;
the concentration of each additive component is as follows: 3-8mg/mL of ferric chloride, 20-30mg/mL of amidoamine, 5-10mg/mL of glucose, 6-10mg/mL of medlar extract, 15-20mg/mL of tomato extract, 5-12mg/mL of vitamin C, 1-3mg/mL of insulin, 12-17mg/mL of olive oil extract and 3-5mg/mL of growth factor.
Preferably, the tomato extract mainly contains glutathione as a required component.
Preferably, the essential component of the olive oil extract is mainly fatty acid.
Preferably, the components required by the medlar extract are mainly procyanidin.
Preferably, the concentration of the additive is 3mg/mL of ferric chloride, 20mg/mL of amidoamine, 5mg/mL of glucose, 6mg/mL of medlar extract, 15mg/mL of tomato extract, 5mg/mL of vitamin C5mg, 1mg/mL of insulin, 12mg/mL of olive oil extract and 3mg/mL of growth factor.
Preferably, the concentration of the additive is 5mg/mL of ferric chloride, 24mg/mL of amidoamine, 7mg/mL of glucose, 8mg/mL of medlar extract, 16mg/mL of tomato extract, 7mg/mL of vitamin C, 7mg/mL of vitamin C, 1.7mg/mL of insulin, 13mg/mL of olive oil extract and 3.7mg/mL of growth factor.
Preferably, the concentration of the additive is 7mg/mL of ferric chloride, 27mg/mL of amidoamine, 8.6mg/mL of glucose, 8.8mg/mL of medlar extract, 18mg/mL of tomato extract, vitamin C9mg/mL, 2.1mg/mL of insulin, 15mg/mL of olive oil extract and 4.3mg/mL of growth factor.
Preferably, the ferric chloride is 8mg/mL, the amidoamine is 30mg/mL, the glucose is 10mg/mL, the medlar extract is 10mg/mL, the tomato extract is 20mg/mL, the vitamin is 12mg/mL, the insulin is 3mg/mL, the olive oil extract is 17mg/mL, and the growth factor is 5 mg/mL.
Preferably, the preparation method of the stem cell culture medium comprises the following steps:
taking part of adipose tissue, cutting, cleaning, placing into a centrifuge for centrifugation at the centrifugation speed of 1500-;
uniformly paving the taken tissue blocks in a culture dish, pouring a culture medium, and culturing by using an incubator, wherein the temperature of the incubator is kept at 37 ℃, and the incubation time is 12 hours;
and thirdly, replacing the original culture dish every 12 hours, abandoning the original culture dish, replacing a new culture dish, inoculating the stem cells into the new culture dish for culture, digesting the stem cells after the stem cells grow to 85-90% of fusion degree, and mixing the stem cells and the new culture dish according to the proportion of 1: 4, passage ratio.
The invention has the beneficial effects that:
1. the additive components used by the stem cell culture medium are ferric chloride, ammonia amide, glucose, a Chinese wolfberry extract, a tomato extract, vitamin C, insulin, an olive oil extract and growth factors, so that the stem cell culture medium does not contain animal serum and is convenient for separating stem cells at the later stage;
2. the culture medium provided by the invention can further improve the culture efficiency of stem cells and reduce the use cost of the culture medium;
3. improve the proliferation activity of the adipose-derived stem cells and the growth capacity of the adipose-derived stem cells.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a stem cell culture medium comprises a substrate culture medium and an additive, wherein the substrate culture medium is a DMEM/F12 liquid culture medium, and the components of the additive added into the substrate culture medium are ferric chloride and ferric chloride which are inorganic salts, and are used for supplementing inorganic salts, ammonia amide, glucose, a Chinese wolfberry extract, a tomato extract, vitamin C, insulin, an olive oil extract and growth factors required in the tissue culture process;
the concentration of each additive component is as follows: 3-8mg/mL of ferric chloride, 20-30mg/mL of amidoamine, 5-10mg/mL of glucose, 6-10mg/mL of medlar extract, 15-20mg/mL of tomato extract, 5-12mg/mL of vitamin C, 1-3mg/mL of insulin, 12-17mg/mL of olive oil extract and 3-5mg/mL of growth factor.
The tomato extract mainly contains glutathione as essential component, and has antioxidant and antidotal effects.
The essential component of olive oil extract is mainly fatty acid, which can provide heat, is a good energy source, and can maintain the relative mobility of cell membrane to ensure the normal physiological function of cells.
The fructus Lycii extract mainly contains procyanidin, and has strong antioxidant and free radical scavenging effects.
The concentration of the additive is 3mg/mL of ferric chloride, 20mg/mL of amidoamine, 5mg/mL of glucose, 6mg/mL of medlar extract, 15mg/mL of tomato extract, 5mg/mL of vitamin C, 1mg/mL of insulin, 12mg/mL of olive oil extract and 3mg/mL of growth factor.
The concentration of the additive is 5mg/mL of ferric chloride, 24mg/mL of amidoamine, 7mg/mL of glucose, 8mg/mL of medlar extract, 16mg/mL of tomato extract, 7mg/mL of vitamin C, 7mg/mL of vitamin C, 1.7mg/mL of insulin, 13mg/mL of olive oil extract and 3.7mg/mL of growth factor.
The concentration of the additive is 7mg/mL of ferric chloride, 27mg/mL of amidoamine, 8.6mg/mL of glucose, 8.8mg/mL of medlar extract, 18mg/mL of tomato extract, 9mg/mL of vitamin C, 2.1mg/mL of insulin, 15mg/mL of olive oil extract and 4.3mg/mL of growth factor.
8mg/mL of ferric chloride, 30mg/mL of amidoamine, 10mg/mL of glucose, 10mg/mL of medlar extract, 20mg/mL of tomato extract, 12mg/mL of vitamin, 3mg/mL of insulin, 17mg/mL of olive oil extract and 5mg/mL of growth factor.
A preparation method of a stem cell culture medium comprises the following steps:
taking part of adipose tissue, cutting, cleaning, placing into a centrifuge for centrifugation at the centrifugation speed of 1500-;
uniformly paving the taken tissue blocks in a culture dish, pouring a culture medium, and culturing by using an incubator, wherein the temperature of the incubator is kept at 37 ℃, and the incubation time is 12 hours;
and thirdly, replacing the original culture dish every 12 hours, abandoning the original culture dish, replacing a new culture dish, inoculating the stem cells into the new culture dish for culture, digesting the stem cells after the stem cells grow to 85-90% of fusion degree, and mixing the stem cells and the new culture dish according to the proportion of 1: 4, passage ratio.
Comparative example 1:
the concentration of the additive is 3mg/mL of ferric chloride, 20mg/mL of amidoamine, 5mg/mL of glucose, 6mg/mL of medlar extract, 15mg/mL of tomato extract, 5mg/mL of vitamin C, 5mg/mL of insulin, 12mg/mL of olive oil extract and 3mg/mL of growth factor;
a preparation method of a stem cell culture medium comprises the following steps:
taking part of adipose tissue, cutting, cleaning, placing into a centrifuge for centrifugation at the centrifugation speed of 1500-;
uniformly paving the taken tissue blocks in a culture dish, pouring a culture medium, and culturing by using an incubator, wherein the temperature of the incubator is kept at 37 ℃, and the incubation time is 12 hours;
and thirdly, replacing the original culture dish every 12 hours, abandoning the original culture dish, replacing a new culture dish, inoculating the stem cells into the new culture dish for culture, digesting the stem cells after the stem cells grow to 85-90% of fusion degree, and mixing the stem cells and the new culture dish according to the proportion of 1: 4, passage ratio.
Example 1:
the concentration of the additive is 3mg/mL of ferric chloride, 20mg/mL of amidoamine, 5mg/mL of glucose, 6mg/mL of medlar extract, 18mg/mL of tomato extract, 5mg/mL of vitamin C, 5mg/mL of insulin, 12mg/mL of olive oil extract and 3mg/mL of growth factor,
a preparation method of a stem cell culture medium comprises the following steps:
taking part of adipose tissue, cutting, cleaning, placing into a centrifuge for centrifugation at the centrifugation speed of 1500-;
uniformly paving the taken tissue blocks in a culture dish, pouring a culture medium, and culturing by using an incubator, wherein the temperature of the incubator is kept at 37 ℃, and the incubation time is 12 hours;
and thirdly, replacing the original culture dish every 12 hours, abandoning the original culture dish, replacing a new culture dish, inoculating the stem cells into the new culture dish for culture, digesting the stem cells after the stem cells grow to 85-90% of fusion degree, and mixing the stem cells and the new culture dish according to the proportion of 1: 4, passage ratio.
Example 2:
the concentration of the additive is 3mg/mL of ferric chloride, 20mg/mL of amidoamine, 5mg/mL of glucose, 6mg/mL of medlar extract, 15mg/mL of tomato extract, 7/7 mg/mL of vitamin C, 1mg/mL of insulin, 12mg/mL of olive oil extract and 3mg/mL of growth factor;
a preparation method of a stem cell culture medium comprises the following steps:
taking part of adipose tissue, cutting, cleaning, placing into a centrifuge for centrifugation at the centrifugation speed of 1500-;
uniformly paving the taken tissue blocks in a culture dish, pouring a culture medium, and culturing by using an incubator, wherein the temperature of the incubator is kept at 37 ℃, and the incubation time is 12 hours;
and thirdly, replacing the original culture dish every 12 hours, abandoning the original culture dish, replacing a new culture dish, inoculating the stem cells into the new culture dish for culture, digesting the stem cells after the stem cells grow to 85-90% of fusion degree, and mixing the stem cells and the new culture dish according to the proportion of 1: 4, passage ratio.
Example 3:
the concentration of the additive is 3mg/mL of ferric chloride, 20mg/mL of amidoamine, 5mg/mL of glucose, 6mg/mL of medlar extract, 15mg/mL of tomato extract, 5mg/mL of vitamin C, 5mg/mL of insulin, 17mg/mL of olive oil extract and 3mg/mL of growth factor;
a preparation method of a stem cell culture medium comprises the following steps:
taking part of adipose tissue, cutting, cleaning, placing into a centrifuge for centrifugation at the centrifugation speed of 1500-;
uniformly paving the taken tissue blocks in a culture dish, pouring a culture medium, and culturing by using an incubator, wherein the temperature of the incubator is kept at 37 ℃, and the incubation time is 12 hours;
and thirdly, replacing the original culture dish every 12 hours, abandoning the original culture dish, replacing a new culture dish, inoculating the stem cells into the new culture dish for culture, digesting the stem cells after the stem cells grow to 85-90% of fusion degree, and mixing the stem cells and the new culture dish according to the proportion of 1: 4, passage ratio.
Example 4:
the concentration of the additive is 3mg/mL of ferric chloride, 20mg/mL of amidoamine, 5mg/mL of glucose, 15mg/mL of tomato extract, 5mg/mL of vitamin C5mg, 1mg/mL of insulin, 12mg/mL of olive oil extract and 3mg/mL of growth factor;
a preparation method of a stem cell culture medium comprises the following steps:
taking part of adipose tissue, cutting, cleaning, placing into a centrifuge for centrifugation at the centrifugation speed of 1500-;
uniformly paving the taken tissue blocks in a culture dish, pouring a culture medium, and culturing by using an incubator, wherein the temperature of the incubator is kept at 37 ℃, and the incubation time is 12 hours;
and thirdly, replacing the original culture dish every 12 hours, abandoning the original culture dish, replacing a new culture dish, inoculating the stem cells into the new culture dish for culture, digesting the stem cells after the stem cells grow to 85-90% of fusion degree, and mixing the stem cells and the new culture dish according to the proportion of 1: 4, passage ratio.
The primary adipose-derived stem cells prepared in the comparative example and examples 1 to 4 were collected by centrifugation, counted by resuspension, and the survival rate was calculated. The results are shown in Table 1.
TABLE 1
From the above, 5.1 × 107-5.9 × 107 primary adipose-derived stem cells are finally obtained from 50ml of tissue, and the survival rate is 98.8-99.4%.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Claims (9)
1. A stem cell culture medium comprises a substrate culture medium and additives, and is characterized in that the substrate culture medium is a DMEM/F12 liquid culture medium, and the additives added into the substrate culture medium comprise ferric chloride, ammonia amide, glucose, a Chinese wolfberry extract, a tomato extract, vitamin C, insulin, an olive oil extract and growth factors;
the concentration of each additive component is as follows: 3-8mg/mL of ferric chloride, 20-30mg/mL of amidoamine, 5-10mg/mL of glucose, 6-10mg/mL of medlar extract, 15-20mg/mL of tomato extract, 5-12mg/mL of vitamin C, 1-3mg/mL of insulin, 12-17mg/mL of olive oil extract and 3-5mg/mL of growth factor.
2. The stem cell culture medium according to claim 1, wherein the tomato extract comprises glutathione as a major component.
3. The stem cell culture medium according to claim 1, wherein the essential component of the olive oil extract is mainly fatty acid.
4. The stem cell culture medium of claim 1, wherein the extract of Lycium barbarum comprises procyanidins as the essential component.
5. The stem cell culture medium according to claim 1, wherein the concentration of the additive is 3mg/mL of ferric chloride, 20mg/mL of amidoamine, 5mg/mL of glucose, 6mg/mL of the extract of Lycium barbarum, 15mg/mL of the extract of tomato, 5mg/mL of vitamin C, 1mg/mL of insulin, 12mg/mL of the extract of olive oil, and 3mg/mL of growth factor.
6. The stem cell culture medium according to claim 1, wherein the concentration of the additive is 5mg/mL of ferric chloride, 24mg/mL of amidoamine, 7mg/mL of glucose, 8mg/mL of the extract of Lycium barbarum, 16mg/mL of the extract of tomato, 7mg/mL of vitamin C, 7mg/mL of insulin, 1.7mg/mL of the extract of olive oil, and 3.7mg/mL of the growth factor.
7. The stem cell culture medium according to claim 1, wherein the concentration of the additive is 7mg/mL of ferric chloride, 27mg/mL of amidoamine, 8.6mg/mL of glucose, 8.8mg/mL of the extract of Lycium barbarum, 18mg/mL of the extract of tomato, 9mg/mL of vitamin C, 2.1mg/mL of insulin, 15mg/mL of the extract of olive oil, and 4.3mg/mL of growth factor.
8. The stem cell culture medium according to claim 1, wherein the ferric chloride is 8mg/mL, the amidoamine is 30mg/mL, the glucose is 10mg/mL, the medlar extract is 10mg/mL, the tomato extract is 20mg/mL, the vitamin is 12mg/mL, the insulin is 3mg/mL, the olive oil extract is 17mg/mL, and the growth factor is 5 mg/mL.
9. A preparation method of a stem cell culture medium is characterized by comprising the following steps:
taking part of adipose tissue, cutting, cleaning, placing into a centrifuge for centrifugation at the centrifugation speed of 1500-;
uniformly paving the taken tissue blocks in a culture dish, pouring a culture medium, and culturing by using an incubator, wherein the temperature of the incubator is kept at 37 ℃, and the incubation time is 12 hours;
and thirdly, replacing the original culture dish every 12 hours, abandoning the original culture dish, replacing a new culture dish, inoculating the stem cells into the new culture dish for culture, digesting the stem cells after the stem cells grow to 85-90% of fusion degree, and mixing the stem cells and the new culture dish according to the proportion of 1: 4, passage ratio.
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